The long-term objective of this training award is to develop an independent research career with emphasis on understanding pathogenesis of M. tuberculosis (MTB). MTB is the leading infectious cause of mortality worldwide. Despite the emergence of multiple drug-resistant strains and lack of a fully effective vaccine against this pathogen, the molecular bases of virulence of MTB are incompletely understood. Given that bacterial gene expression is reflective of their environment and expression of many virulence-associated genes is tightly regulated, we hypothesize that in vivo expression profiling will offer a rational approach to identifying and studying genes that contribute to MTB virulence and persistence. The immediate goal of the proposed studies is to use a combination of techniques to determine the role of MTB homologs of M. marinum granuloma-activated genes (gags) in pathogenesis of MTB in a mouse model. In our preliminary studies, we have used RT-PCR to identify MTB putative gags (p-gags) that are expressed in infected mouse lungs, and have used quantitative RT-PCR to identify and confirm those p-gags that are expressed at higher levels in the lungs of mice than in broth culture. To determine the role of in vivo induced p-gags in pathogenesis, we are currently making p-gag deletion mutants with conditionally replicating mycobacteriophages and will test them for attenuation in mice. To characterize and understand the function of selected genes, we will use transgenic mice to determine which cell subsets and cytokines trigger the in vivo induction of these genes. Finally, given that MTB p-gags likely represent only a small subset of MTB genes expressed late in infection, we will develop and validate tools for comprehensive profiling of MTB in vivo gene expression with microarrays. The proposed studies could have important clinical implications. The co-sponsorship of this award offers a unique environment where I will receive the technical and professional training needed to become an independent investigator.